Racl、NF-κB、IL-1β在DVT形成中表达变化的研究
摘要
旧的]
1.采用基因芯片检测大鼠DVT模型股静脉壁组织中差异表达的基因,查阅Ncbi PubMed和GeneCard数据库分析基因功能,研究静脉壁中Rac1、IL-1β等基因的mRNA表达变化与DVT形成的关系。
2.采用彩色多普勒超声动态监测兔DVT模型的静脉血栓形成状态以便更准确采集静脉血液样本,并应用PCR筛查差异表达基因,研究兔血细胞中Rac1、 IL-1β等基因的mRNA表达变化与DVT形成之间的关系。
3.采用Ncbi BLAST比较大鼠、兔、人Rac1、IL-1β等基因的mRNA序列相似度,推测Rac1、IL-1β等基因在人DVT形成中可能具有重要作用；
4.采用RCR筛查Rac1、IL-1β等基因在DVT患者血细胞中的mRNA表达变化,运用real-time PCR确定Rac1等差异表达的基因,研究人血细胞中Rac1、IL-1β等基因的mRNA表达变化与DVT形成的关系；
[材料方法]
第一部分：大鼠DVT模型股静脉壁组织基因芯片检测,筛选差异表达基因
将67只SD大鼠随机分为对照组(10只)和模型组(57只),对模型组采用蚊式钳钳夹双侧股静脉联合石膏制动双下肢构建大鼠DVT模型。选取不同时间点(造模后2.5h和25h)处死大鼠,解剖双侧股静脉观测：血栓的发生率、严重程度,进一步将模型组分为：血栓形成前组10只(造模后2.5h取材)、血栓形成组和血栓不形成组(共45只,造模后25h取材,依据单侧或双侧股静脉中是否有血栓形成分组)。获取股静脉壁组织,采用Affymetrix Rat230A cDNA基因芯片检测差异表达的基因；采用倍数变化分析法(Fold Change, FC)筛选出显著差异表达的基因；查阅最新Ncbi PubMed和GeneCard数据库,筛选出Rac1、 IL-1β等可能与血栓形成、活性氧(ROS)生成高度相关的基因；
第二部分：PCR筛查Rac1等基因在兔DVT模型血细胞中的表达化
将18只日本大耳兔随即分为假手术组(5只)和模型组(13只),采用结扎双侧股总静脉、股深静脉构建兔DVT模型。在不同的时间点(造模前Od、造模后1d、造模后2d、造模后3d、造模后8d),采用彩色多普勒超声准确监测双侧股总静脉、股深静脉、股浅静脉血栓形成状态,并采集血液样本,采用PCR检测静脉血细胞中Rac1、IL-1β等基因mRNA的表达变化。
第三部分：Rac1、IL-1β等基因在大鼠、兔与人之间的同源性分析
查阅Ncbi Gene和GeneCard数据库确定大鼠、兔的Rac1、IL-1β等基因的mRNA序列,通过BLAST与人类相应基因进行序列比较,同源性分析Rac1等基因在大鼠、兔和人之间的mRNA序列相似度；推测Rac1、IL-1β等基因在DVT形成中可能具有重要作用；
第四部分：PCR及real-time PCR检测DVT患者血细胞中Racl等基因的mRNA表达变化
参考《静脉血栓栓塞症预防的NICE指南》(2012年)和《美国胸科医师协会抗栓与血栓预防临床实践指南—深静脉血栓形成的诊断》(2012年第9版)制定本研究的DVT诊断标准：DVT的综合诊断=危险因素评估(基础危险因素+专科危险因素)+主要临床表现+下肢深静脉彩色多普勒超声检查(附录2)。纳入研究对象排除其它高危因素(怀孕、糖尿病、肿瘤、免疫系统疾病等)后,于2010年3月-2012年3月共纳入研究对象58例,正常对照组(20例,为健康志愿者)、血栓未形成组(20例,为下肢长骨骨折或脊柱骨折7d内行手术后无形成血栓的患者)、血栓形成组(18例,为DVT形成患者)。三组均采集清晨空腹静脉血液样本,血栓未形成组于手术后第1d采集,血栓形成组于确诊后第1d采集。采用PCR筛查Rac1、IL-1β等基因在DVT患者血细胞中的1mRNA表达变化；进一步采用real-time PCR确定差异表达的基因；并查Ncbi PubMed和GeneCard数据库分析Rac1、NF-κB1、IL-1β之间的相互调控关系,及其对内皮细胞、白细胞、血小板功能的影响。
[结果]
1.大鼠DVT股静脉内皮基因芯片检测结果：造模后2.5小时,大鼠股静脉壁组织中Rac1、Rac2、NF-κB1、IL-1β的荧光强度明显增加,血栓形成前组明显高于对照组；造模后25小时,继续显著增加,血栓形成组高于对照组、血栓形成前组和血栓不形成组,而对照组与血栓不形成组间没有差异；
2.彩色多普勒监测兔DVT模型股总静脉、股深静脉、股浅静脉血流信号结果：造模后1d,50%以上的静脉开始形成血栓；造模后2d,80%以上的静脉无血流信号,血栓形成达高峰；造模后3d,开始有部分静脉出现断续血流信号,血栓开始发生机化；造模后8d,50%以上的静脉出现断续血流信号,大部分静脉开始机化再通;
3.PCR筛查兔DVT模型血细胞中差异表达基因的结果：模型组Rac1、NF-κB2、 IL-1β的mRNA表达水平,在造模后1d开始明显上调,并持续维持到造模后2d、3d、8d,均明显高于造模前(0d)及假手术组各个时间点(0d、1d、2d、3d、8d)(P<0.05)；在造模后1d、2d、3d、8d之间的差异没有统计学意义(P>0.05)；而Rac2mRNA的表达在两组各个时间点间的差异均没有统计学意义(P>0.05)
4.大鼠、兔、人Rac1等基因mRNA序列的BLAST结果：大鼠Rac1、Rac2、 NF-κB1、NF-κB2、IL-1β基因与人类相应基因序列的相似度分别为80%、78%、96%、92%、78%；兔Rac1、Rac2、NF-κB2、IL-1β基因与人类相应基因序列的相似度分别为72%、93%、99%、68%。
5.DVT患者血细胞中的PCR筛查及real-time PCR检测结果：PCR筛查发现Rac2、Rac3、NF-κB2mRNA的表达在三组之间的差异没有统计学意义(P>0.05);real-time PCR检测结果与PCR筛查结果一致,均发现Rac1、 NF-κB1、IL-1βmRNA的表达在血栓形成组中明显高于血栓未形成组和对照组(P<0.05),而血栓未形成组和正常对照组之间的差异没有统计学意义(P>0.05)。
[结论]
1.大鼠DVT模型股静脉内皮中Rac1、NF-κB1和IL-1β的mRNA表达上调可能与DVT的形成密切相关；
2.彩色多普勒超声可准确动态监测兔DVT模型深静脉血栓形成状态；
3.兔血细胞中Rac1、NF-κB2和IL-1βmRNA表达上调可能与DVT形成密切相关；
4.人血细胞中Rac1、NF-κB1和IL-1βmRNA表达上调可能与DVT形成高度相关；
Expression of Racl, NF-κB and IL-1β in deep venous thrombosis ABSTRACT
[Objective]
1. Applying gene chip to screen the differential expressed genes in femoral vein wall tissue of rats DVT model, and searching the Ncbi gene and GeneCard database to explore relationship between Racl, NF-κB1, IL-1β mRNA expression change gene and DVT.
2. Using color doppler ultrasound to dynamic monitor thrombosis state of rabbit DVT model, accuratly collecting blood samples, and adopting PCR to screen these genes expression in blood of rabbits DVT model, and exploring the relationship between differential expression genes and DVT.
3. Through homology analyzing these gene sequences similarity between rats/rabbits and human and consulting literatures, then speculating that these genes may play an important role DVT;
4. Applying RCR to detetct these genes expression in DVT patient blood cell, furthermore, make use of real-time RCR to identify these genes expression, so as to explore relationship between these genes mRNA expresseion DVT.
[Materials and Methods]
Part1:Screening the differential epressed genes in femoral vein wall tissue of rats DVT model by gene chip
The67rats were randomly divided into control group (non-modeling, n=10) and model group (n=57). Rat DVT model was established by combination with clamping both femoral veins and fixing with cast in model group. The incidence and serious degree of thrombus were observed by dissecting rat femoral vein in different time points (2.5hours and25hours after modeling). Model group were further divided into pre-thrombogenesis group (harvested venous tissue at2.5hours after modeling), thrombogenesis group and non-thrombogenesis group (harvested venous tissue at25hours after modeling, and grouped according to whether thrombosis). Acquiring the femoral vein wall tissue and extracting total RNA. Applying the Genechip Rat Genome2302.0to detect genes expression and using FC (Fold Change) to analyse the differential expression genes from experimental data. Searching the Ncbi gene and GeneCard database so as to find out Racl, NF-κB1and IL-1β closely related to ROS (Reactive Oxygen Species) production.
Part2:Detecting these genes expression in blood of rabbits DVT model by PCR
The involved18rabbits were randomly divided into sham group (non-modeling, n=5) and model group (n=13). Rabbit DVT model was established by ligaturing bilateral common femoral vein and deep femoral vein. Applying color doppler ultrasound to accurately monitor the thrombosis status of common femoral vein, deep femoral vein and superficial femoral vein, and collecting the blood sample. Adoptting the PCR technology to detecte expression changes of these genes in blood sample, including Racl, IL-1β and so on. Part3:Homology analysis of these gene between rats/rabbits and human
Consulting Ncbi gene and GeneCard database to acertain these gene sequences of rats and rabbits, through homology analyzing these gene sequences similarity between rats/rabbits and human and consulting literatures.
Part4:Adoptting PCR and real-time PCR to detecte theses genes expression changes in blood sample of DVT patients
Reference "venous thromboembolism prevention NICE guide"(2012) and Diagnosis of DVT:Antithrombotic Therapy and Prevention of Thrombosis,9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (2012,9th version)", formulate the DVT diagnosis standard of this study:DVT comprehensive diagnosis=dangerous factor assessment (foundation risk factors+specialized subject risk factors)+major clinical manifestations+color doppler ultrasound (appendix2). Ruling out other risk factors (pregnancy, diabetes, cancer, immune system disease, and so on), included58research objects were normal control group (20cases, healthy volunteers), non-thrombogenesis group (20cases for lower limb fractures, long bone or spine fracture undergo operation within7day, and without thrombosis after surgery), thrombosis group (18cases DVT patients), during march2010to march2012. Fasting vein blood samples of three groups were collected in early morning. Non-thrombogenesis group was1day after surgery. Thrombosis group was1day after diagnosis. These genes expression changes (including Rac1, NF-κB1, IL-1β, etc) were screened by PCR. The differential expression genes were identified by the real-time. Futhermore, the mutual regulation relationship among Rac1, NF-κB1and IL-1β, and the function influnence on endothelial cell, leucocyte cell and platelet, were analyzed by Ncibi PuMed and GeneCard, so as to explore these genes role in regulating DVT.
[Results]
1. The test results of gene chip in rats DVT model femoral venous wall found that: The expressions of Rac1, Rac2, NF-κB1and IL-1(3in rat femoral venous wall tissue were significantly up-regulated at2.5hours after modeling (pre-thrombogenesis group was higher than control group), and continued up-regulating at25hours after modeling (thrombogenesis group was higher than the pre-thrombogenesis group, non-thrombogenesis group and control group). But the difference in three groups was not detected.
2. The results of adopting color doppler to monitor blood flow signal of common femoral vein, deep femoral vein and superficial femoral vein in rabbit DVT model found that:more than50%of vein have thrombosis at Id after modeling; more than80%of the vein have no blood flow signal at2d after modeling, indicated that venous thrombosis reached peak; Part of the vein began to appear intermittently flow signals at3d after modeling, seggested that venous thrombosis started to organizing; More than50%of the vein appeared intermittently flow signals at8d after modeling, suggest that most of the venous thrombosis emerged organization and recanalization.
3. The results of applying PCR to screen differential expression gene in blood samples of rabbit DVT model found that:the mRNA expressions of Rac1, NF-κB2, and IL-1β were significantly up-regulated at1d after modeling and continues to maintain2d,3d,8d after modeling, were higher than Od before modeling of model group and different time points (0d、1d、2d、3d、8d) of sham group (P<0.05); The difference at different time points (1d、2d、3d、8d after modeling) was not deteceted (P>0.05); The Rac2mRNA expressions difference at different time points of two group was not deteceted (P>0.05).
4. The BLAST results of Rac1etc gene sequences between rats/rabbits and human found that:Rac1, Rac2, NF-κB1, NF-κB2and IL-1β gene sequences similarity between rats and human respectively were80%,78%,96%,92%and78%; Rac1, Rac2, NF-κB2and IL-1(3gene sequences similarity between rabbits and human respectively were72%、93%、99%、68%.
5. The test results of PCR in blood samples of DVT patients found that:The difference of the mRNA expressions of Rac2, Rac3and NF-κB2was not detected in three groups (P>0.05); The results of real-time PCR and PCR showed that the mRNA expressions of Rac1, NF-κB1and IL-1β in thrombogenesis group was significantly higher than the non-thrombogenesis group and control group (P<0.05), and the difference was not detected between nonthrombogenesis group and control group (P>0.05)
[Conclusion]
1. Upregulated expression of Rac1, NF-κB1and IL-1β in rats DVT modle femoral vein wall tissue closely related to DVT.
2. The color doppler ultrasound can accurate dynamic monitoring thrombosis state of the rabbit DVT model;
3. Upregulated expression of Rac1、NF-κB2and IL-1β in rabbits DVT modle blood closely crosponded with the progress of thrombosis, and might be played regulated role in DVT.
4. Upregulated expression of Rac1、NF-κB1and IL-1β in blood sample of DVT patienet closely related to DVT.
1.采用基因芯片检测大鼠DVT模型股静脉壁组织中差异表达的基因,查阅Ncbi PubMed和GeneCard数据库分析基因功能,研究静脉壁中Rac1、IL-1β等基因的mRNA表达变化与DVT形成的关系。
2.采用彩色多普勒超声动态监测兔DVT模型的静脉血栓形成状态以便更准确采集静脉血液样本,并应用PCR筛查差异表达基因,研究兔血细胞中Rac1、 IL-1β等基因的mRNA表达变化与DVT形成之间的关系。
3.采用Ncbi BLAST比较大鼠、兔、人Rac1、IL-1β等基因的mRNA序列相似度,推测Rac1、IL-1β等基因在人DVT形成中可能具有重要作用；
4.采用RCR筛查Rac1、IL-1β等基因在DVT患者血细胞中的mRNA表达变化,运用real-time PCR确定Rac1等差异表达的基因,研究人血细胞中Rac1、IL-1β等基因的mRNA表达变化与DVT形成的关系；
[材料方法]
第一部分：大鼠DVT模型股静脉壁组织基因芯片检测,筛选差异表达基因
将67只SD大鼠随机分为对照组(10只)和模型组(57只),对模型组采用蚊式钳钳夹双侧股静脉联合石膏制动双下肢构建大鼠DVT模型。选取不同时间点(造模后2.5h和25h)处死大鼠,解剖双侧股静脉观测：血栓的发生率、严重程度,进一步将模型组分为：血栓形成前组10只(造模后2.5h取材)、血栓形成组和血栓不形成组(共45只,造模后25h取材,依据单侧或双侧股静脉中是否有血栓形成分组)。获取股静脉壁组织,采用Affymetrix Rat230A cDNA基因芯片检测差异表达的基因；采用倍数变化分析法(Fold Change, FC)筛选出显著差异表达的基因；查阅最新Ncbi PubMed和GeneCard数据库,筛选出Rac1、 IL-1β等可能与血栓形成、活性氧(ROS)生成高度相关的基因；
第二部分：PCR筛查Rac1等基因在兔DVT模型血细胞中的表达化
将18只日本大耳兔随即分为假手术组(5只)和模型组(13只),采用结扎双侧股总静脉、股深静脉构建兔DVT模型。在不同的时间点(造模前Od、造模后1d、造模后2d、造模后3d、造模后8d),采用彩色多普勒超声准确监测双侧股总静脉、股深静脉、股浅静脉血栓形成状态,并采集血液样本,采用PCR检测静脉血细胞中Rac1、IL-1β等基因mRNA的表达变化。
第三部分：Rac1、IL-1β等基因在大鼠、兔与人之间的同源性分析
查阅Ncbi Gene和GeneCard数据库确定大鼠、兔的Rac1、IL-1β等基因的mRNA序列,通过BLAST与人类相应基因进行序列比较,同源性分析Rac1等基因在大鼠、兔和人之间的mRNA序列相似度；推测Rac1、IL-1β等基因在DVT形成中可能具有重要作用；
第四部分：PCR及real-time PCR检测DVT患者血细胞中Racl等基因的mRNA表达变化
参考《静脉血栓栓塞症预防的NICE指南》(2012年)和《美国胸科医师协会抗栓与血栓预防临床实践指南—深静脉血栓形成的诊断》(2012年第9版)制定本研究的DVT诊断标准：DVT的综合诊断=危险因素评估(基础危险因素+专科危险因素)+主要临床表现+下肢深静脉彩色多普勒超声检查(附录2)。纳入研究对象排除其它高危因素(怀孕、糖尿病、肿瘤、免疫系统疾病等)后,于2010年3月-2012年3月共纳入研究对象58例,正常对照组(20例,为健康志愿者)、血栓未形成组(20例,为下肢长骨骨折或脊柱骨折7d内行手术后无形成血栓的患者)、血栓形成组(18例,为DVT形成患者)。三组均采集清晨空腹静脉血液样本,血栓未形成组于手术后第1d采集,血栓形成组于确诊后第1d采集。采用PCR筛查Rac1、IL-1β等基因在DVT患者血细胞中的1mRNA表达变化；进一步采用real-time PCR确定差异表达的基因；并查Ncbi PubMed和GeneCard数据库分析Rac1、NF-κB1、IL-1β之间的相互调控关系,及其对内皮细胞、白细胞、血小板功能的影响。
[结果]
1.大鼠DVT股静脉内皮基因芯片检测结果：造模后2.5小时,大鼠股静脉壁组织中Rac1、Rac2、NF-κB1、IL-1β的荧光强度明显增加,血栓形成前组明显高于对照组；造模后25小时,继续显著增加,血栓形成组高于对照组、血栓形成前组和血栓不形成组,而对照组与血栓不形成组间没有差异；
2.彩色多普勒监测兔DVT模型股总静脉、股深静脉、股浅静脉血流信号结果：造模后1d,50%以上的静脉开始形成血栓；造模后2d,80%以上的静脉无血流信号,血栓形成达高峰；造模后3d,开始有部分静脉出现断续血流信号,血栓开始发生机化；造模后8d,50%以上的静脉出现断续血流信号,大部分静脉开始机化再通;
3.PCR筛查兔DVT模型血细胞中差异表达基因的结果：模型组Rac1、NF-κB2、 IL-1β的mRNA表达水平,在造模后1d开始明显上调,并持续维持到造模后2d、3d、8d,均明显高于造模前(0d)及假手术组各个时间点(0d、1d、2d、3d、8d)(P<0.05)；在造模后1d、2d、3d、8d之间的差异没有统计学意义(P>0.05)；而Rac2mRNA的表达在两组各个时间点间的差异均没有统计学意义(P>0.05)
4.大鼠、兔、人Rac1等基因mRNA序列的BLAST结果：大鼠Rac1、Rac2、 NF-κB1、NF-κB2、IL-1β基因与人类相应基因序列的相似度分别为80%、78%、96%、92%、78%；兔Rac1、Rac2、NF-κB2、IL-1β基因与人类相应基因序列的相似度分别为72%、93%、99%、68%。
5.DVT患者血细胞中的PCR筛查及real-time PCR检测结果：PCR筛查发现Rac2、Rac3、NF-κB2mRNA的表达在三组之间的差异没有统计学意义(P>0.05);real-time PCR检测结果与PCR筛查结果一致,均发现Rac1、 NF-κB1、IL-1βmRNA的表达在血栓形成组中明显高于血栓未形成组和对照组(P<0.05),而血栓未形成组和正常对照组之间的差异没有统计学意义(P>0.05)。
[结论]
1.大鼠DVT模型股静脉内皮中Rac1、NF-κB1和IL-1β的mRNA表达上调可能与DVT的形成密切相关；
2.彩色多普勒超声可准确动态监测兔DVT模型深静脉血栓形成状态；
3.兔血细胞中Rac1、NF-κB2和IL-1βmRNA表达上调可能与DVT形成密切相关；
4.人血细胞中Rac1、NF-κB1和IL-1βmRNA表达上调可能与DVT形成高度相关；
Expression of Racl, NF-κB and IL-1β in deep venous thrombosis ABSTRACT
[Objective]
1. Applying gene chip to screen the differential expressed genes in femoral vein wall tissue of rats DVT model, and searching the Ncbi gene and GeneCard database to explore relationship between Racl, NF-κB1, IL-1β mRNA expression change gene and DVT.
2. Using color doppler ultrasound to dynamic monitor thrombosis state of rabbit DVT model, accuratly collecting blood samples, and adopting PCR to screen these genes expression in blood of rabbits DVT model, and exploring the relationship between differential expression genes and DVT.
3. Through homology analyzing these gene sequences similarity between rats/rabbits and human and consulting literatures, then speculating that these genes may play an important role DVT;
4. Applying RCR to detetct these genes expression in DVT patient blood cell, furthermore, make use of real-time RCR to identify these genes expression, so as to explore relationship between these genes mRNA expresseion DVT.
[Materials and Methods]
Part1:Screening the differential epressed genes in femoral vein wall tissue of rats DVT model by gene chip
The67rats were randomly divided into control group (non-modeling, n=10) and model group (n=57). Rat DVT model was established by combination with clamping both femoral veins and fixing with cast in model group. The incidence and serious degree of thrombus were observed by dissecting rat femoral vein in different time points (2.5hours and25hours after modeling). Model group were further divided into pre-thrombogenesis group (harvested venous tissue at2.5hours after modeling), thrombogenesis group and non-thrombogenesis group (harvested venous tissue at25hours after modeling, and grouped according to whether thrombosis). Acquiring the femoral vein wall tissue and extracting total RNA. Applying the Genechip Rat Genome2302.0to detect genes expression and using FC (Fold Change) to analyse the differential expression genes from experimental data. Searching the Ncbi gene and GeneCard database so as to find out Racl, NF-κB1and IL-1β closely related to ROS (Reactive Oxygen Species) production.
Part2:Detecting these genes expression in blood of rabbits DVT model by PCR
The involved18rabbits were randomly divided into sham group (non-modeling, n=5) and model group (n=13). Rabbit DVT model was established by ligaturing bilateral common femoral vein and deep femoral vein. Applying color doppler ultrasound to accurately monitor the thrombosis status of common femoral vein, deep femoral vein and superficial femoral vein, and collecting the blood sample. Adoptting the PCR technology to detecte expression changes of these genes in blood sample, including Racl, IL-1β and so on. Part3:Homology analysis of these gene between rats/rabbits and human
Consulting Ncbi gene and GeneCard database to acertain these gene sequences of rats and rabbits, through homology analyzing these gene sequences similarity between rats/rabbits and human and consulting literatures.
Part4:Adoptting PCR and real-time PCR to detecte theses genes expression changes in blood sample of DVT patients
Reference "venous thromboembolism prevention NICE guide"(2012) and Diagnosis of DVT:Antithrombotic Therapy and Prevention of Thrombosis,9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (2012,9th version)", formulate the DVT diagnosis standard of this study:DVT comprehensive diagnosis=dangerous factor assessment (foundation risk factors+specialized subject risk factors)+major clinical manifestations+color doppler ultrasound (appendix2). Ruling out other risk factors (pregnancy, diabetes, cancer, immune system disease, and so on), included58research objects were normal control group (20cases, healthy volunteers), non-thrombogenesis group (20cases for lower limb fractures, long bone or spine fracture undergo operation within7day, and without thrombosis after surgery), thrombosis group (18cases DVT patients), during march2010to march2012. Fasting vein blood samples of three groups were collected in early morning. Non-thrombogenesis group was1day after surgery. Thrombosis group was1day after diagnosis. These genes expression changes (including Rac1, NF-κB1, IL-1β, etc) were screened by PCR. The differential expression genes were identified by the real-time. Futhermore, the mutual regulation relationship among Rac1, NF-κB1and IL-1β, and the function influnence on endothelial cell, leucocyte cell and platelet, were analyzed by Ncibi PuMed and GeneCard, so as to explore these genes role in regulating DVT.
[Results]
1. The test results of gene chip in rats DVT model femoral venous wall found that: The expressions of Rac1, Rac2, NF-κB1and IL-1(3in rat femoral venous wall tissue were significantly up-regulated at2.5hours after modeling (pre-thrombogenesis group was higher than control group), and continued up-regulating at25hours after modeling (thrombogenesis group was higher than the pre-thrombogenesis group, non-thrombogenesis group and control group). But the difference in three groups was not detected.
2. The results of adopting color doppler to monitor blood flow signal of common femoral vein, deep femoral vein and superficial femoral vein in rabbit DVT model found that:more than50%of vein have thrombosis at Id after modeling; more than80%of the vein have no blood flow signal at2d after modeling, indicated that venous thrombosis reached peak; Part of the vein began to appear intermittently flow signals at3d after modeling, seggested that venous thrombosis started to organizing; More than50%of the vein appeared intermittently flow signals at8d after modeling, suggest that most of the venous thrombosis emerged organization and recanalization.
3. The results of applying PCR to screen differential expression gene in blood samples of rabbit DVT model found that:the mRNA expressions of Rac1, NF-κB2, and IL-1β were significantly up-regulated at1d after modeling and continues to maintain2d,3d,8d after modeling, were higher than Od before modeling of model group and different time points (0d、1d、2d、3d、8d) of sham group (P<0.05); The difference at different time points (1d、2d、3d、8d after modeling) was not deteceted (P>0.05); The Rac2mRNA expressions difference at different time points of two group was not deteceted (P>0.05).
4. The BLAST results of Rac1etc gene sequences between rats/rabbits and human found that:Rac1, Rac2, NF-κB1, NF-κB2and IL-1β gene sequences similarity between rats and human respectively were80%,78%,96%,92%and78%; Rac1, Rac2, NF-κB2and IL-1(3gene sequences similarity between rabbits and human respectively were72%、93%、99%、68%.
5. The test results of PCR in blood samples of DVT patients found that:The difference of the mRNA expressions of Rac2, Rac3and NF-κB2was not detected in three groups (P>0.05); The results of real-time PCR and PCR showed that the mRNA expressions of Rac1, NF-κB1and IL-1β in thrombogenesis group was significantly higher than the non-thrombogenesis group and control group (P<0.05), and the difference was not detected between nonthrombogenesis group and control group (P>0.05)
[Conclusion]
1. Upregulated expression of Rac1, NF-κB1and IL-1β in rats DVT modle femoral vein wall tissue closely related to DVT.
2. The color doppler ultrasound can accurate dynamic monitoring thrombosis state of the rabbit DVT model;
3. Upregulated expression of Rac1、NF-κB2and IL-1β in rabbits DVT modle blood closely crosponded with the progress of thrombosis, and might be played regulated role in DVT.
4. Upregulated expression of Rac1、NF-κB1and IL-1β in blood sample of DVT patienet closely related to DVT.
引文
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